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Sci Rep. 2017 Jan 19;7:40948. doi: 10.1038/srep40948.

Time-resolved neutron scattering provides new insight into protein substrate processing by a AAA+ unfoldase.

Ibrahim Z1,2,3,4, Martel A4, Moulin M4, Kim HS1,2,3, Härtlein M4, Franzetti B1,2,3, Gabel F1,2,3,4.

Author information

1
Université Grenoble Alpes, Institut de Biologie Structurale, 38044 Grenoble, France.
2
Centre National de la Recherche Scientifique, Institut de Biologie Structurale, 38044 Grenoble, France.
3
Centre à l'Energie Atomique et aux Energies Alternatives, Institut de Biologie Structurale, 38044 Grenoble, France.
4
Institut Laue-Langevin, 38042 Grenoble, France.

Abstract

We present a combination of small-angle neutron scattering, deuterium labelling and contrast variation, temperature activation and fluorescence spectroscopy as a novel approach to obtain time-resolved, structural data individually from macromolecular complexes and their substrates during active biochemical reactions. The approach allowed us to monitor the mechanical unfolding of a green fluorescent protein model substrate by the archaeal AAA+ PAN unfoldase on the sub-minute time scale. Concomitant with the unfolding of its substrate, the PAN complex underwent an energy-dependent transition from a relaxed to a contracted conformation, followed by a slower expansion to its initial state at the end of the reaction. The results support a model in which AAA ATPases unfold their substrates in a reversible power stroke mechanism involving several subunits and demonstrate the general utility of this time-resolved approach for studying the structural molecular kinetics of multiple protein remodelling complexes and their substrates on the sub-minute time scale.

PMID:
28102317
PMCID:
PMC5244417
DOI:
10.1038/srep40948
[Indexed for MEDLINE]
Free PMC Article

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